Description of the Prior Art
A wide variety of allergenic extracts and methods for preparing, storing, and using them including standard commercial procedures are described in Remington's Pharmaceutical Sciences, p. 1344-1352, Mack, Easton, Pennsylvania, 15 ed. (1975). Allergenic extracts from a wide range of natural and plant and animal sources and manufactured goods are described. Manufacturing procedures for preparing extracts including details about grinding, defatting, extraction, clarification, dialysis, concentration, sterilization and lyophilization are presented therein.
Major efforts to concentrate and standardize allergenic components of allergen extracts have been made in the past. Procedures for solvent extraction, often combined with precipitation, are described in U.S. Pat. Nos. 2,316,311, 3,148,122, 3,281,323, 3,591,677, 3,953,588, 3,995,023, 4,027,006, and 2,347,435. Ion exchange techniques are described in 2,901,398. The principal objective of these processes is to separate the active allergenic components from inactive ingredients obtained through the particular extraction procedure employed.
A number of patents have been directed to the preparation of modified allergenic compounds wherein the allergenic component is chemically modified such as by cross-linking with formaldehyde or other reactive chemical agent. As a preliminary procedure, the allergenic extracts may be separated from low molecular weight components which would interfere with the chemical reaction. Removal of lower molecular weight components for this purpose is described in U.S. Pat. No. 4,226,853 by (dialysis), 4,234,569 (ultrafiltration or gel filtration), 4,256,732 (dialysis), and 4,163,778 (dialysis and column chromatography). In the above procedures, the aqueous or ethanolic extract solution is treated to remove the lower molecular weight components, and the chemical reactants are added to the aqueous solution of the product to effect the chemical modification of the allergenic protein.
Of a similar nature are the papers by E. Puttonen et al, "Studies on Allergen and Allergoid Preparations for Purified Timothy (Phleum pratense) Pollen Extracts" in Int. Arch. Allergy Appl. Immun. 68:1-12 (1982). In this paper, fractionation of a dried timothy pollen extract by gel column chromatography is presented. In the procedure, a sample of the extract was fractionated with a SEPHADEX G-75 column, and active fractions were pooled and freeze-dried. One portion of the material was then reacted with formaldehyde to prepare an allergoid product, and the properties of the original and treated fractions are compared.
The freeze-dried timothy grass pollen intermediates described in the Puttonen et al article are completely different from the extracts of this invention. In the sampling of active fractions for pooling, minor allergens are discarded, and the relative ratios of allergens present in the original extract are not preserved. The freeze-dried product cannot therefore be correlated with diagnostic or desensitizing compositions which are based on the natural allergen composition profile. This publication does not describe separation of plant extracts using ultrafiltration or lowering the moisture content of the resultant solution to less than one weight percent to increase the shelf life stability thereof.
Stability studies of timothy grass pollen extracts are disclosed by M.C. Anderson et al in "Antigenic and Allergenic Change During Storage of a Pollen Extract", J. Allergy Clin. Immunol. 69(1) pp 3-10 (1982) and articles cited therein. Enzyme activities of timothy pollen extracts are reported, and degradation mechanisms including thermal denaturation and enzymatic breakdown of allergenic and antigenic proteins and carbohydrates is postulated. Sugar-splitting activity was reported to be inhibited in 50 percent glycerol solutions.